The long-term objective of this project is to understand the molecular mechanisms responsible for altered glucose homeostasis during highly active antiretroviral therapy. The studies in this proposal are intended to identify the cellular targets of HIV protease inhibitors that lead to impaired beta-cell function and alterations in hepatic glucose production and to elucidate the molecular mechanism of this inhibition. We hypothesize that the peptide structure within all currently available HIV protease inhibitors is responsible for acute and reversible inhibition of the insulin-responsive glucose transporter GLUT4 and the liver/pancreas transporter GLUT2. To test this hypothesis, the acute effects of HIV protease inhibitors on the glucose-stimulated insulin secretory pathway in freshly isolated rodent and human islets as well as cultured MIN6 cells will be determined. Euglycemic hyperinsulinemic clamp experiments will also be performed in rats to determine the acute effects of HIV protease inhibitors on hepatic glucose production. The ability of the biguanide metformin to prevent protease inhibitor mediated inhibition of GLUT4 activity will be tested in Xenopus oocytes heterologously expressing this transporter isoform. Finally, the structural determinants involved in PI-induced GLUT4 inhibition will be determined by testing a family of synthetic aromatic peptides for their ability to inhibit 2-deoxyglucose uptake in GLUT4 expressing oocytes. Taken together, these studies will provide new insights into the molecular mechanism(s) leading to insulin resistance in patients treated with HIV protease inhibitors. This may facilitate the design of newer HIV protease inhibitors that maintain their clinical efficacy while avoiding their adverse effects on glucose homeostasis and will assist efforts to develop more effective treatment strategies.